Expression of tetanus toxin fragment C in yeast

ABSTRACT

Expression of tetanus toxin fragment C is accomplished employing a DNA coding sequence having a (G+C)-content that has been increased in the region from nucleotide 410 to the 3&#39; end of the coding sequence relative to the wild-type DNA sequence. This allows the production of complete mRNA transcripts. Typically the (G+C)-content is increased in the following regions: (i) nucleotides 510-710, (ii) nucleotides 650-850, (iii) nucleotides 800-1100, (iv) nucleotides 900-1200 and (v) nucleotides 1100 to the 3&#39; end of the coding sequence. The (G+C)-content may also be increased in the region of nucleotides 410-610. These regions in the wild-type DNA encompass terminator sequences.

The present invention relates to the production of tetanus toxin Cfragment.

Vaccination against tetanus is effective in the prevention of thisdisease in most Western countries, although incomplete vaccination insome third world countries can account for up to one million cases oftetanus every year. Current tetanus vaccines are produced byformaldehyde treatment of tetanus toxin produced by the anaerobicbacterium C. tetani to produce the immunogenic toxoid. It has beensuggested that impurities incorporated during formaldehyde treatment arepartly responsible for the adverse effects sometimes seen withhyperimmunisation with tetanus toxoid.

The structural gene for tetanus toxin has been cloned and sequenced(Fairweather, N. F., et al, J. Bacteriol. 165, 21-27 (1986);Fairweather, N. F., and Lyness, V. A., Nuc. Acid Res. 14, 7809-7812(1986). These studies have confirmed the structure of tetanus toxin as a150kD protein of 1315 amino acids. The toxin can be cleaved by varioustreatments into several fragments. Fragment C, comprising the C terminal451 amino acids, is a 50kD polypeptide generated by papain cleavage oftoxin.

Fragment C derived in this way has been shown to be non-toxic and iscapable of immunising mice and guinea pigs (Helting, T. B., and Zwisler,O., J. Biol. Chem. 252, 187-193 (1977); Helting, T. B., and Nau, H. H.,Act. Pathol. Microbiol. Scan. Sect. C 92, 59-63 (1984)). Papaindigestion also releases the 100 kD fragment B, comprising the N-terminalpart of the toxin molecule. Fragment B is also protective, but has beenreported to be toxic to animals at high doses (Helting, T. B., et al, J.Biol. Chem. 253,125-125, (1978)).

Portions of tetanus toxin containing fragment C have been expressed inE.coli (Fairweather, N. F., et al, J. Bacteriol, 165, 21-27, (1986));

Fairweather, N. F., et al, Infection and Immunity 55, 2541-2545, (1987);EP-A-0209281). These portions of tetanus toxin which were expressed wereeither fused to part of the E.coli trpE protein or comprised part offragment B and all of fragment C of tetanus toxin. All the above werefound to be expressed at low levels and were all insoluble in thecytoplasm of E.coli cells.

It has been found previously that when fragment C on its own isexpressed in E.coli, it is soluble in the cytoplasm of the cells.Fragment C was expressed using two plasmids, pTETtac1 and pTETtac2 whichwere derived from the high expressing plasmid pIFGtac124A (Makoff, A.J., et al., Biochem. Soc. Trans., 16, 48-49, (1988)) Most of the codingsequence of pTETtac1 was provided by two restriction fragments. The restof the sequence was encoded by a pair of synthetic oligonucleotides both42 base pairs long, where the codon bias was optimised for expression inE.coli. Plasmid pTETtac2 was constructed from pTETtac1 by replacing theBglIIoSfa NI region by a pair of synthetic oligonucleotides (each 161nucleotides long) which reproduced the sequence upstream of theinitiation codon and optimised the coding sequence, at the beginning ofthe C fragment region, for expression in E.coli (Makoff, A. J., et al.Bio/Technology 7, 1043-1046 (1989)).

However, E.coli has the disadvantage as a host organism that it containstoxic pyrogenic factors (lipopolysaccharides from the cell wall) whichmust be rigorously excluded from the final product. The ease with whichthese factors may be excluded will depend on the protein product inquestion and the method by which it is purified from the cell. However,it would be preferable to eliminate the possibility of contaminationaltogether simply by using a non-toxic organism as the host, such asyeast.

In using the native sequence encoding fragment C, the inventors wereunable to obtain expression in yeast and found that the barrier toexpression was due to the fact that the mRNA transcripts of the genewere incomplete. Synthesis of the complete transcript probably involvesat the 3'-end three closely linked steps: termination of the primarytranscript, endonucleolytic processing and polyadenylation. (Platt,J.,Ann. Rev. Biochem., 55, 339-372, (1986)). The inventors have nowidentified the position of several "terminators" (termination/endo-nucleolytic processing/polyadenylation sites) present in the DNA. As aresult the inventors were able to eliminate these and obtain successfulexpression in yeast of tetanus toxin fragment C.

FIG. 1 shows the position of at least six elements which are completelyor partially responsible for the production of incomplete mRNAtranscripts. The yeast terminator is poorly defined. Several differentconsensus sequences have been proposed (Henikoff, S., et al., Cell, 33,607-614, (1983); Zaret, K. S., and Sherman, F., Cell, 28, 563-573,(1982); Bennetzen, J. L., and Hall, B. D., J.Biol. Chem., 257,3018-3025, (1982a)), but it appears that there may be deviation fromthese sequences and it appears that other, undefined elements may alsobe necessary for termination (Osborne, B. I., and Guarente L., PNAS, 86,4097-4101, (1989)). Yeast terminators occur in stretches of (A+T)-richDNA, though not all (A+T)-rich DNA contains terminators. Our surprisingfinding was that the original fragment C DNA contained at least sixelements which were responsible for incomplete transcription of themRNA. The elements were eliminated by increasing the (G+C)-content atthese positions thus providing for the production of a substantiallycomplete mRNA transcript.

The present invention provides a novel DNA sequence encoding tetanustoxin fragment C and having a (G+C)-content that has been increasedrelative to the wild-type DNA sequence so as to allow the production ofcomplete mRNA transcripts in yeast.

Tetanus toxin fragment C, as used herein, is defined as the wild typepolypeptide having the amino acid sequence set forth in FIG. 2 and inSeq ID Nos: 1 and 2 or is a mutant polypeptide having an amino acidsequence that is at least 90% homologous with that set forth in FIG. 2and that retains substantially the same biological and immunogenicproperties as the wild-type polypeptide.

The amino acid sequence of fragment C may be varied by one or more aminoacid substitutions, extensions, insertions and/or deletions provided theresulting polypeptide retains substantially the same biological andimmunogenic properties as wild-type fragment C.

In the case of amino acid substitutions, one or more of the amino acidresidues of fragment C may be replaced by one or more other amino acidresidues which achieve this aim. Candidate substitutions include Ser forThr and vice versa, Glu for Asp and vice versa, Gln for Asn and viceversa, Leu for Ile and vice versa, Ala for Val and vice versa and Argfor Lys and vice versa.

Mutant fragment C may be obtained by introducing nucleotide changes intothe DNA sequence encoding wild-type fragment C, for example into the DNAsequence of FIG. 2 and SEQ ID No: 1. This may be achieved by anyappropriate technique, including restriction of the sequence with anendonuclease, insertion of oligonucleotide linkers, use of anexonuclease and/or a polymerase and site-directed mutagenesis.

Fragment C wild-type DNA has a (G+C) - content of 29%, while thepreferred DNA sequence in accordance with the present invention (seeFIG. 2 and SEQ ID NO: 3) has 47%. The maximum possible (G+C) - contentthat can encode fragment C is 60%. A level of 40-60% (G+C)- contentwould thus allow the production of a complete mRNA transcript providedthat were no localised concentrations of (A+T) rich DNA.

In designing a fragment C gene for expression in yeast, one route wouldbe to use codons found in highly expressed yeast genes (Bennetzen, J.L., and Hall, B. D., J.Biol. Chem., 257, 3026-3031, (1982)) This wouldincrease the (G+C)-content. Another important consideration would be toeliminate runs of (A+T) since these would raise the local (A+T)-contentand might be sufficient to cause termination.

Since the elements responsible for the production of incompletetranscripts are only likely to extend over approximately 100nucleotides, it is possible to achieve the same result by onlyincreasing the (G+C)-content within these small regions.

Six regions were identified as being responsible for the incompleteproduction of mRNA transcripts by analysis of a number of differentmutant DNA sequences containing differing lengths of DNA for which the(G+C)-content had been increased.

                  TABLE 1                                                         ______________________________________                                        Region responsible for                                                        production of incomplete                                                                         Region to be altered so as                                 transcript in C. tetani DNA                                                                      to allow the production of                                 (nucleotides into coding sequence)                                                               complete mRNA transcripts                                  ______________________________________                                        1. 560 ± 50     410-610                                                    2. 660 ± 50     510-710                                                    3. 800 ± 50     650-850                                                    4. 1000 ± 100    800-1100                                                  5. 1100 ± 100    900-1200                                                  6. 1300 ± 100   1100-1400                                                  ______________________________________                                    

It is believed that some of the regions are more responsible than othersfor the production of incomplete transcripts. It appears that regions 2and 4 are most important. In order to allow the production of completemRNA transcripts which is being prevented by regions 2 and 4 the(G+C)-content of mutant fragment DNA is increased relative to the nativeDNA sequence from nucleotide 510 to nucleotide 700 and from nucleotide800 to nucleotide 1100, the numbers corresponding to those set forth inthe sequence of FIG. 2 and in SEQ ID Nos: 1 and 3. The next mostimportant regions are 3,5 and 6. Similarly, in order to allow theproduction of complete mRNA transcripts which are additionally beingprevented by regions 3, 5 and 6 the (G+C)-content is additionallyincreased from nucleotide 650 to nucleotide 850, from nucleotide 900 tonucleotide 1200 and from nucleotide 1100 to nucleotide 1400, the numberscorresponding to those set forth in the sequence of FIG. 2. Region 1 maybe too weak to interfere with the production of complete mRNAtranscripts; however, in order to allow complete mRNA production whichis being prevented by Region 1 the (G+C)-content is additionallyincreased from nucleotide 410 to nucleotide 610, the numberscorresponding to those set forth in the sequence of FIG. 2.

It can be seen from Table 1 that because of the clustering of elements,it is advisable to increase the (G+C)-content from nucleotide 410 to the3'- end nucleotide, the numbers corresponding to those set forth in thesequence of FIG. 2 so as to allow the production of complete mRNAtranscripts.

The novel DNA sequence according to the invention may be chemicallysynthesised and cloned using methodologies well-known in the art.

The novel DNA may then be cloned into a suitable vector and used totransform yeast which is then capable of expressing the polypeptidewhich is encoded by the novel DNA.

The vector may be any appropriate vector which is suitable for thecloning of the DNA and which may be used to transform a yeast cell andthereby express the relevant protein. Such vectors include autonomouslyreplicating plasmids and chromosomal integration vectors.

Vectors which may be used for cloning DNA include pWYG7 (see Example 1and FIG. 3), pWYG5 (see Example 2 and FIG. 5) and PIC3 (Example 6) foruse in yeast.

In yet another feature of the present invention there is provided anexpression vector, which incorporates a DNA sequence according to theinvention and which is capable of expressing fragment C in yeast (SeeExamples 4 and 5).

The expression vector incorporates control elements for transcriptionalinitiation (promoters) and termination. The coding sequence of the geneto be expressed along with its own translational start and stop codonsis inserted between these control elements.

Examples of promoters for use with the expression vector of the presentinvention include GAL1, GAL7, ADH2, PGK, GAPDH, etc. (Kingsman, S. M. etal., Biotechnology & Genetic Engineering Reviews, Vol. 3, 377-416,(1985); Russell, D. W. et al., The Journal of Biological Chemistry, Vol258, No.4, 2674-2682 (1983)); and AOX1 (Digam, et al., Dev. Ind. Micro.Biol, 29, 59-65, (MS8)). Use of the inducible promoter such as the GALl,GAL7 or ADH2 promoter may be preferred as it enables expression to becontrolled. Expression of the GAL1 and GAL7 promoters is induced bygalactose.

An appropriate expression vector may be obtained by cloning a DNAsequence according to the present invention into an expression vector.An example of a complete expression vector, containing the GAL1promoter, is pWYG5-TET15 which contains the whole synthesised DNAencoding fragment C (see FIG. 12).

In a further aspect of the invention there is provided a yeast organismtransformed with an expression vector according to the invention.

Examples of suitable host cells for use in the above-described methodare yeast cells such as Saccharomyces, Pichia, Kluyveromyces orHansenula and in particular the following species; Saccharomycescerevisiae, Kluyveromyces lactis, Hansenula polymorpha, or Pichiapastoris.

A strain of yeast which can be used is Saccharomyces cerevisiae strainS150-2B.

The present invention provides a process for the preparation of fragmentC of tetanus toxin which process comprises the steps of:

(i) preparing the DNA of fragment C to contain codons of increased(G+C)-content by chemically synthesising the entire coding sequence

(ii) inserting the DNA into a suitable vector

(iii)transforming yeast cells

(iv) culturing a transformed host to express fragment C of tetanus toxin

(v) recovering the product fragment C thus expressed

Recombinant tetanus toxin fragment C may therefore be obtained thusfacilitating its use as the basis for an alternative vaccine toformaldehyde treated tetanus toxoid and tetanus toxin fragment C asexpressed in E.coli.

Step (iv) of the process of the invention comprises culturing yeasttransformed by the expression vector of the present invention such as tocause expression of fragment C. Fragment C may then be isolated from theyeast cells by for example breaking the yeast cells with glass beads orwhen the material is secreted by isolation from the culture medium.

The DNA sequence and corresponding amino acid sequence encoded byplasmid pWYG5-TET15 mentioned below is shown in FIG. 2 and in SEQ IDNos: 3 and 4. The symbol , , , is shown under the translational stopcodon. The nucleotide changes made in the synthesised gene are shownbelow the original C.tetani DNA sequence.

The fragment C that is expressed is recovered, in step (v) of thepresent process, from the yeast cells by similar protocols by standardpurification procedures. (Makoff, A. J., et al., Bio/Technology, 7,1043-1046, (1989a)).

The fragment C may be isolated to the desired degree of purity. Someminor yeast contaminants may also be present. Generally the degree ofpurity is at least 80%, preferably at least 90% and more preferably atleast 95%.

The present invention also provides a vaccine for conferring immunity totetanus comprising tetanus toxin fragment C prepared according to theinvention and a pharmaceutically acceptable carrier or diluent. Thevaccine may include other antigens to provide a multi-valent vaccine.Typically carriers and diluents are sterile, pyrogen-free liquid mediasuitable as vehicles for introducing a polypeptide into a patient.Isotonic saline solution may be employed.

The vaccine may also comprise an adjuvant for stimulating the immuneresponse and thereby enhancing the effect of the vaccine. A convenientadjuvant is aluminium hydroxide. Conveniently the vaccines areformulated to contain a final concentration of fragment C or itsderivative of from 0.2 to 200 μg/ml, preferably 5 to 50 μg/ml, mostpreferably about 30μg/ml. After formulation the vaccine may beincorporated into a sterile container which is then sealed and stored ata low temperature, for example 4° C., or it may be freeze-dried.

The vaccine may be administered by any conventional method for theadministration of vaccines such as parenteral (e.g. subcutaneous orintramuscular) injection. The treatment may consist of a single dose ofvaccine or a plurality of doses over a period of time. It is recommendedthat each dose is 0.1 to 2ml preferably 0.2 to 1 ml, most preferablyabout 0.5ml of vaccine.

The inventors have surprisingly found that it is possible to secretefragment C into the culture medium using an appropriate secretion signalsuch as the alpha factor leader peptide. The protein was found to besecreted to a level of 5-10mg/l into the medium and was present in twoforms in roughly equal amounts: a high molecular mass hyper-glycosylatedprotein (75-200kDa), and a core-glycosylated protein (65kDa). Thisglycosylated protein was found to be substantially inactive invaccinating mice against tetanus toxin. However, if the glycosylatedprotein is de-glycosylated it becomes as active as the intracellularfragment C in immunising against tetanus.

As it should be possible to secrete fragment C to levels in excess of100mg/l in high-density fermentations the de-glycosylated secretedproduct may provide a feasible production alternative to theintracellular protein production.

The invention will be described in more detail hereinafter withreference to the accompanying drawings in which:

FIG. 1 shows the location of elements responsible for the production ofincomplete transcripts identified in the four variants of fragment C DNAhaving different amounts of synthesised DNA. Coding regions for fragmentC are boxed; regions that were chemically synthesised with codonsoptimal for translation in E.coli are hatched. The four versions of thegene, TET2, TET7, TET11 and TET15, had 12%, 50%, 73% and 99% syntheticDNA, respectively. The approximate positions of yeast polyadenylationsites found in the native sequence, estimated from the sizes of shorttranscripts in Northern blots, are indicated by arrows. (The 5'synthesised region in TET2 extends 16Ont into the gene, and the firstterminator is at 560±5Ont).

FIG. 2 shows the sequence of C.tetant DNA encoding fragment C (topline), the nucleotide changes made in the fully synthesised version offragment C (middle line) and the amino acid sequence (third line) (SEQ.ID Nos: 1 to 4).

FIG. 3 shows the construction of the yeast expression vector pWYG 7.Foreign genes are inserted between the Bam HI and Bcl I sites.

FIG. 4 shows the nucleotide sequence of the promoter region of GAL7 (SEQID No: 5). The synthesised promoter corresponds to the XhoI to BamHIfragment. Regions downstream of BamH1 are present in native GAL7including the RNA start site (↓) and the initiating ATG (underlined).The two basepairs which were altered to give a BamHI site areunderlined.

FIGS. 5 (i)-5(ii) shows the construction of yeast expression vectorpWYG5.

FIG. 6 shows the map of pTETtac2

FIG. 7 shows the E.coli vector for expression of tetanus toxin fragmentC (pTETtac2) with progressively more synthesised DNA containing optimalcodons. Only the region between the EcoRI and AvaI sites is shown, thefull map of pTETtac2 being given in FIG. 6. The fragment C codingregions are boxed and synthesised regions are hatched.

FIG. 8(i)-8(iii) shows the construction of pTETtac16. Theoligonucleotides inserted into pTETtac7 to obtain pTETtac14 are shown inSEQ ID NOS: 6 and 7. FIG. 9i)-9(ii) shows the construction ofpWYG7-TET2. The oligonucleotides inserted into pTETtac2 to obtainpTETtac2Y are shown in SEQ ID NOS: 8 and 9.

FIG. 10 shows a Western blot analysis of proteins from induced cellscontaining no plasmid, pWYG7-TET2, pWYG5-TET7, pWYG5-TET11 orpWYG5-TET15 (tracks 1 to 5, respectively). Track 6 was loaded withMet-fragment C produced in E coli. The proteins (50μg) were separated ina 9% SDS-polyacrylamide gel, blotted onto nitrocellulose, and probedwith a rabbit anti-fragment C serum as first antibody. Track 3 containsa very faint doublet at about 30kDa which is not visible in thereproduction.

FIG. 11 shows a Northern blot of RNA extracted from induced cellstransformed with pWYG7-TET2, pWYG5-TET7, pWYG5-TET11 and pWYG5-TET15(tracks 1 to 4, respectively). The position of stained RNA size markers(size in kb) is indicated. The blot was probed with ³² P-labelled 1.4kbBglII-BamHI fragment of pTETtac2.

FIG. 12 shows the map of pWYG5-TET15

FIG. 13 shows the nucleotide sequence of the synthetic DNA fragmentscarrying the α-factor prepro region used in pWYG69-TET2 and pWYG59-TET15(SEQ ID NOS: 10 and 11).

FIG. 14 shows a Western blot of secreted yeast fragment C. Lane 1,pWY659-TET15 culture supernatant treated with endoglycosidase H. Lanes 2and 3, untreated pWY659-TET15 culture supernatant. Lane 4, pWYG9-TET2culture supernatant. Lane 5, pWY69-TET2 culture supernatant treated withendoglycosidase H. Lane 6, culture supernatant from untransformed cells.Lane 7, culture supernatant from untransformed cells afterendoglycosidase H treatment. Lane 8, molecular weight markers. Lane 9,fragment C produced in E.coli.

FIG. 15(a)-15(b) shows the construction of pP1C3-TET15. Theoligonucleotides inserted in pA0804 to obtain pPIC1 are shown in SEQ IDNOS: 12 and 13.

FIG. 16(a)-16(b) shows fragment C production in different pP1C3-TET15transformants. Part a) shows proteins from total cell extracts separatedon a Coomassie blue stained SDS-polyacrylamide gel. Lanes 1-11 areloaded with extracts from clones 885C, 887C, 8811C, 8812D, 881D, 882E,885E, 8811E, 881F, 8810F, 883H respectively. Lane 12, extract fromfragment C expressing E.coli. Lane 13, molecular weight markers(phosphorylase b, 97,400; bovine serum albumin, 68,000; ovalbumin,43,000; chymotrypsinogen, 25,700; lactoglobulin, 18,400). Lane 14,insoluble fraction from 881F. Lane 15, total extract from 881F. Lane 16,soluble fraction from 881F. Part b) shows a Western blot of thesesamples. Lanes 1-9, as in part a). Lane 10, extract from 889F. Lane 11,extract from 8810F. Lane 12, extract from 883H. Lane 13, extract fromuntransformed cells. Lane 14, molecular weight markers.

FIG. 17 shows a Coomassie blue stained SDS polyacrylamide gel showingfragment C production in a high cell density fermentation of clone 881F.Lane 1, molecular weight markers (β-galactosidase, 116,000;phosphorylase b, 97,400; bovine serum albumin, 68,000; ovalbumin,43,000; carbonic anhydrase, 29,000). Lane 2, untransformed cell extract.Lane 3, 881F extract from an induced shake-flask culture. Lanes 4-14,extracts from cells taken from the fermenter at the following timeintervals with respect to the beginning of induction,-15,0,2,4,6,8,24,28,30,32,52 hours.

The following Examples illustrate the present invention and are notintended to limit the invention in any way.

EXAMPLE 1

1. Construction of yeast expression vector pWYG7

The vector pWYG7, (Beesley, K. M., et al., Bio/Technology, 8, 644-649(1990)), constructed at Wellcome, was used for the expression of Cfragment. The construction of pWYG7 is outlined in FIG. 3. It is derivedfrom the 2u vector pJDB219 (Beggs, J. D., Nature, 275, 104-109, (1978))modified to contain a kanamycin-resistance marker (kan^(r)) and theyeast galactose-regulated GAL7 promoter. First the kan^(r) marker(HincII fragment from pUC4K; Vieira, J., and Messing, J., Gene, 19, 259,(1982)) was ligated into the unique SmaI site of pJDB219 to give thekan^(r) tet^(r) vector pJDB219K. Secondly, a synthetic GAL7 promoterfragment (XhoI-BamHI fragment, sequence shown in FIG. 4 and in SEQ IDNO: 5) was cloned between the unique SalI and BamHI sites of pJDB219K.The resulting vector, pWYG7, has the GAL7 promoter with unique BamHI andBclI sites upstream of the yeast 2u plasmid FLP gene transcriptionalterminator (Sutton, A., and Broach, J. R., Mol.Cell. Biol, 5, 2770-2780(1985)). Foreign genes to be expressed from pWYG7 are inserted betweenthe BamHI and BclI sites. The design of the GAL7 promoter fragment isdiscussed below.

The smallest fragment of DNA upstream of the GAL7 gene which exhibitsfull promoter activity has been defined by deletion mapping (Tajima, M.,et al., Yeast, 1, 67-77, (1985)). Based on this information a 260bp GAL7promoter fragment was synthesised (FIG. 4 for sequence). The 260bppromoter was synthesised as four overlapping oligonucleotides using aPharmacia Gene Assembler (protocol supplied by Pharmacia). Theseoligonucleotides were phosphorylated and annealed using standardtechniques, then ligated into XhoI-BamHI cut plC-20H (Marsh, J. C.,Gene, 32,481-485, (1984)). Positive clones were identified and their DNAsequenced using the double-stranded DNA sequencing method with universaland reverse sequencing primers (Hong, G. F., Biosc, Reports, 2, 907,(1982)). The sequence of the GAL7 inserts was confirmed, and then theXhoI-BamHI GAL7 insert was excised and cloned into pJDB219K as describedabove.

The design of the GAL7 promoter fragment in pWYG7 is such that thenatural GAL7 DNA sequence has been slightly modified (2bp changed) inorder to make the BamHI cloning site Upstream of the GAL7 mRNA startsites. The foreign gene to be expressed is then linked with syntheticDNA to the BamHI site, such that the GAL7 mRNA start sites areintroduced, along with the GAL7 upstream untranslated sequences. Thusthe first non-yeast DNA downstream of the promoter is the initiating ATGcodon of the foreign gene, and the transcript produced will have a yeastGAL7 leader rather than a foreign leader which could reduce efficiencyof translations.

EXAMPLE 2

Construction of yeast expression vector pWYG5

The vector pWYG5 is the same basic plasmid as pWYG7 but has the GAL1promoter from pBM150 (Johnston, M. and Davis, R. W. Mol. Cell. Biol 4,1440-1448 (1984)) in place of the GAL7 promoter. The 0.7kb EcoRI-BamHIfragment from pBM150, containing the divergent GAL and GAL10 promoters,was first sub-cloned between the EcoRI and BamHI sites of pIC-20H (Marshet al., J. C., Gene, 32, 481-485, (1984)) to give pIC-GAL, then the0.7kb XhoI-BamHI promoter fragment from pIC-GAL was isolated and placedbetween the SalI and BamHI sites of pJDB219K to give pWYG5 (theconstruction is outlined in FIG. 5).

The GAL1 promoter from pBM150 has a BamHI linker placed downstream ofthe RNA initiation sites and therefore pWYG5 is used differently frompWYG7. Foreign genes must be adapted to have a BamH1 or BamHI-compatible(i.e. BglII or BclI) site immediately upstream of the initiation codon.In order to conform with the consensus found in highly expressed yeastgenes, the sequence upstream of the ATG should be rich in A residues andparticularly have A at -3. As with pWYG7, the foreign genes are insertedbetween the BamHI and BclI sites of pWYG5.

EXAMPLE 3

Construction of E.coli expression vectors for tetanus toxin fragment C,including synthesised versions of the gene, and intermediate vectors foryeast expression.

Expression cassettes of fragment C DNA for transfer to the yeast vectorspWYG5 and pWYG7 were isolated from the E.coli expression vector pTETtac2and its derivatives (Makoff et al., 1989; U.K. patent application No.89141220.0,Ser. No. 07/777,337, filed Nov. 29, 1991. pTETtac2 is a tacpromoter vector containing DNA coding for Met-fragment C (FIG. 6 forplasmid map); the first 161bp and last 42bp of the natural C.tetani DNAhave been replaced by synthesised DNA which was altered to optimisecodon usage for E.coli and to provide useful restriction sites. (Allsynthetic DNA was chemically synthesised as oligonucleotides of length50-160, on a Pharmacia Gene Assembler, which were phosphorylated,annealed and assembled into the relevant plasmids). Expression vectorsbased on pTETtac2 were then constructed where progressively more of theC.tetani DNA, starting from the 5' end, was replaced by synthesised DNAwhose codon usage was optimised for E.coli. The first vector pTETtac7,was constructed via the intermediate plasmid pTETtac 6, shown in FIG. 7;pTETtac7 contains an approximately 45% synthesised gene. This involvedcloning two oligonucleotides between the BanI and MaeII sites ofpTETtac2 in order to produce the two unique sites NcoI and AfIII inpTETtac6. Eight more oligonucleotides were then cloned between the twosites to generate pTETtac11, which contained a 75% synthesised gene.

A version of pTETtac2 containing the 99% synthesised gene (pTETtac15)for fragment C was actually first designed specifically as anintermediate vector (pTETtac16) for transfer of the expression cassetteto the yeast vector pWYG5. The nucleotide sequence of the synthesisedgene is compared to the original C.tetani gene in FIG. 2 from thissequence and the restriction maps in FIG. 7 the sequence of each versionof the gene can be derived. The overall scheme for the construction ofpTETtac16 is shown in FIG. 8. First, pTETtac7 was modified byreplacement of the DNA between the BglII and SalI sites witholigonucleotides which provided upstream sequences compatible with theyeast vector pWYG5 (sequence of oligonucleotides in FIG. 8 and in SEQ IDNOS: 6 and 7). Secondly, the remaining 400bp of the DNA encodingfragment C was synthesised as four oligonucleotides of length 140 to160. These were phosphorylated, annealed and cloned between the ClaI andBamHI sites of pIC-20H. Recombinant plasmids containing the 400bp insertwere identified and further checked by sub-cloning into M13 andsequencing (Sanger. F., et al., Proc.Nat.Acad. Sci., 74, 5463-5467,(1977)). A plasmid with an insert of the correct sequence, designatedpIC-TET, was used as a source of the 400bp ClaI-BamHI fragment to ligateto the 4199bp AfIII-BamHI fragment of pTETtac14 and the 325bp AflII-ClaIfragment of pTETtac11 in order to create pTETtac16. pTETtac16 then hasthe fully synthesised gene for fragment C with codons optimised forE,coli and considerably more (GC)-rich DNA than the C.tetani DNA,preceded by an upstream region suitable for expression in pWYG5.

EXAMPLE 4

Construction of yeast intracellular expression vectors for fragment C.

Four vectors were constructed, one based on pWYG7 and three on pWYG5.The pWYG7 vector, pWYG7-TET2, contained the largely unaltered form ofthe natural C.tetani gene from pTETtac2. The remaining vectors,pWYG5-TET7, pWYG5-TET11 and pWYG5-TET15 were all based on pWYG5 andcontained the genes with progressively more synthesised DNA, from theplasmids pTETtac7, pTETtac11 and pTETtac16, respectively. VectorpWYG5-TET15 was deposited on 16th Feb. 1993 under the Budapest Treaty atthe National Collection of Industrial and Marine Bacteria, Aberdeen,United Kingdom under accession number NCIMB 40538.

(i) pWYG7-TET2

The DNA between the BglII and SalI sites of pTETtac2 was replaced by twooligonucleotides to give pTETtac2Y in order to provide the GAL7 upstreamsequences required for expression in pWYG7 (FIG. 9 for construction andsequences; sequences also shown in SEQ ID NOS: 8 and 9). Theoligonucleotides also placed an NcoI (CCATGG) site at the initiatingATG, altering the second codon from Lys to Val. The 1.4kb BglII-BamHIfragment from pTETtac2Y was isolated and ligated with pWYG7 (dam-DNA)which had been digested with the BamHI and bclI and then with calfintestinal alkaline phosphatase. Recombinant plasmids with inserts ofthe correct orientation were designated pWYG7-TET2.

Western blot analysis of protein extracts from induced cells containingpWYG7-TET2 gave no detectable product reacting with the antibody (track2, FIG. 10). An ELISA quantitation gave an exceedingly low, butpositive, figure of less than 10⁻³ of soluble protein. Since the genefor fragment C was found to be efficiently expressed in a number ofother host cells, the plasmid and transformants were rechecked andexpression re-analysed extensively.

A gene encoding an unmodified fragment C was next tested.

(ii) pWYG5-TET7 and pWYG5-TET11

These plasmids were made by transferring the 1.4kb BglII-BamHI fragmentsfrom pTETtac7 and pTETtac11 into pWYG5, between the BamHI to BclI sites.The transcripts produced by pWYG5-TET7 and pWYG5-TET11 in yeast may betranslated sub-optimally since the upstream regions between the BgllIsite and the initiation codon are designed for E,coli expression, and donot conform to the consensus for highly expressed yeast genes.

Western blot analysis of the products from induced cells containing theplasmid pWYG5-TET7 showed the presence of two faint bands atapproximately 29kDa and 30kDa (track 3, FIG. 10 - too faint to see inreproduction), but no full length fragment C (approximately 50kDa).

This result provided the clue that incomplete transcripts were beingproduced, therefore the fragment C-specific mRNA from pWYG7-TET2 andpWYG7-TET7 was analysed. The Northern blot (FIG. 11) showed that insteadof a full-length transcript (expected size approximately 1655nucleotides), pWYG7-TET2 gave rise to a major band of approximately 700nt and a minor band of 600 nt and pWYG7-TET7 to two bands ofapproximately 900 and 1100 nt. Since these RNAs all hybridised to aprobe from the 5' end of the gene (BglII to NcoI fragment frompTETtac7), incomplete transcripts were being produced within the genefor fragment C. The fact that the transcripts from pWYG-TET7 are largersuggests that the original C.tetani DNA contained sequences which do notallow the production of complete mRNA transcripts, and that these weredestroyed as the DNA was synthesised. This idea is reinforced by thefact that the approximate position of the elements involved with theproduction of an incomplete mRNA transcript in pWYG7 -TET2 is within theregion which was modified by synthesis in pWYG5-TET7.

Induced cells containing pWYG5-TET11 were shown to produce fragment C ofthe correct size on Western blots (FIG. 10), at a concentration ofapproximately0.5% of cell protein. The product was soluble in that itremained in the supernatant after centrifugation at 10,000 g for 15 min.

An analysis of RNA from pWYG5-TET11 (FIG. 11) showed that there were twomajor transcripts of less than full length (1200 and 1400 nt), and onlya minor amount of the full length transcript (approximately 1700 nt).Thus efficient expression was still being prevented by the remaining400bp of C.tetani DNA in the pWYG5-TET11 plasmid.

(iii) pWYG5-TET15

The 1.4kb BglII-BamHI fragment of pTETtac16 was isolated and clonedbetween the BamHI and BclI sites of pWYG5 as described above.

Plasmids with inserts of the correct orientation were designatedpWYG5-TET15 (FIG. 12).

Cells containing this plasmid produced greater amounts of fragment Cthan before, to a level of 2.5% of cell protein (ELISA quantitation andsee FIG. 10). Analysis of the RNA indicated that for the first time mostof the fragment C-specific RNA was full length (FIG. 11). Thus it mustbe concluded from the RNA analysis that the C.tetani DNA encodingfragment C contains at least six elements which are fully or partiallyresponsible for the production of incomplete transcripts in yeast. Thepositions of the elements are shown in FIG. 1. With the present state ofknowledge about transcription of mRNA in yeast few if any of these couldbe predicted from the sequence of the DNA, and they could be removed byre-synthesising the DNA to have a higher (G+C)-content. Alternatively,the elements could be accurately delineated by mapping of the 3' ends ofthe truncated transcripts described above, and only those regionsidentified as being responsible for the production of incompletetranscripts being resynthesised.

EXAMPLE 5

Construction of yeast secretion vectors for fragment C

Two vectors were constructed for the secretion of fragment C, pWYG9-TET2and pWYG59-TET15. These both contained DNA encoding the prepro leaderpeptide from the yeast mating pheromone, alpha-factor (Kurjan, J. andHerskowitz, I., Cell, 30, 933-948, (1982)).

(i) pWYG9-TET2

This vector is similar to pWYG7-TET2 but contains the coding region forthe alpha-factor leader peptide between the BamHI site of the GAL7promoter and the NcoI site at the initiating ATG codon of fragment C.The synthetic DNA fragment contains altered codons, in order to generatea XhoI restriction site to facilitate cloning, giving a conservativeamino acid change (Asp to Glu) immediately upstream of the KEX2 cleavagesite. GAL7 upstream sequences required for expression in pWYG7 are alsoincluded (FIG. 13).

In Western blots of culture supernatants from cells transformed withpWYG9-TET2 a broad smear of reactive material of heterogenous molecularweight (75-200kD) was observed. When de-glycosylated withendoglycosidase H the molecular weight of this was substantially reducedto a major species of approximately 26kD (FIG. 14). This result gavefurther support for the notion that the wild-type C.tetani fragment Cgene contains sequences fortuitously recognised as being responsible forthe production of incomplete mRNA transcripts. The size of this band isconsistent with it being a run-off translation product of the majortranscript characterised by Northern analysis (Example 4).

(ii) pWYG59-TET15

This vector is similar to pWYG5oTET15 but contains the alpha-factorleader peptide coding region between the BamHI site of the GAL1 promoterand the SalI site near the 5' end of the fragment C gene. The syntheticDNA fragment also contains the same NcoI site found at the initiator ATGof pWYG7-TET2 (see FIG. 13).

Two forms of fragment C were found to be secreted into the medium bycells containing pWYG59-TET15. A diffuse band of high molecular weightmaterial was detected similar to that seen with pWYG9-TET2. In addition,a major band of about 65kD was detected (FIG. 14). A ladder of at leastfour other less intense bands of lower molecular weight was alsovisible. All of these species were reduced to approximately 50kD, thesize expected for correctly processed full length fragment C, whentreated with Endo H suggesting that the differences between them are dueto differences in N-linked glycosylation. Fragment C contains sevenpotential sites for the addition of asparagine-linked carbohydrate andour data suggests that at least five of these are actually being usedduring alpha-factor signal directed secretion.

Secretion of full length fragment C by yeast cells containing theresynthesised TET15 gene was found to be efficient. The total amount offragment C secreted to the medium by unoptimised shake flask cultureswas estimated to be about 7μg/ml and none was detected in intracellularextracts from these cultures.

EXAMPLE 6

Construction of Pichia pastoris intracellular expression vectors forfragment C.

The vector pPIC3-TET15, which is derived from pA0804, was used forintracellular expression of fragment C in Pichia (Diagan, et al., Dev.Ind. Microbiol., 29, 59-65, (1988); Sreekrishna et al., Biochemistry,28, 4117-4125 (1989)). This vector uses the promoter from the AOX1 geneto drive expression and can be integrated into the host chromosomal AOX1locus.

To facilitate insertion of the fragment C gene the synthetic adapteroligonucleotides shown in FIG. 15 and in SEQ ID NOS: 12 and 13 werecloned between the AsuII and EcoRI sites of pA0804, to give pPIC1. Aderivative of this plasmid, pPIC2, which lacks the EcoRI site was thenconstructed. This was done by digesting with EcoRI followed by fillingin of the protruding single stranded ends with the Klenow fragment ofDNA polymerase I and the blunt ends were then ligated together. The1.4kb BglII-NheI fragment from pTETtac16 containing the fragment C genewas then inserted between the BamH1 and SpeI sites of pPIC2 to givepPIC3-TET15 as shown in FIG. 15.

Fragment C production in shake flasks, by several pPIC3-TET15transformants that grew slowly on methanol, was examined. FIG. 16 showsSDS-PAGE and Western blotting analysis of cell lysates. Expressionlevels were estimated by densitometric scanning of Coomassie bluestained gels and by ELISA and these varied between differenttransformants from 0.3% of total cell protein to about 11%. Even at thehighest level of expression the product was soluble. The highestexpressing strain, 881F, was used in high cell density inductions in afermenter. Cells were grown to a density of 90g/l (dry weight) beforeinduction. A time course for the induction is shown in FIG. 17.Production of fragment C began rapidly upon induction, rose to a levelof about 20-28% of total cell protein after 24 hrs and remained at thislevel up to 52 hrs after induction. The final level of fragment C in thefermenter was estimated to be about 11g/l and again the product wassoluble.

EXAMPLE 7

Transformation of yeast with fragment C expression vectors

The vectors were introduced into the Saccharomyces cerevisae strainS150-2B (a leu2 his3 ura3 trpl; (McCleod, M., et al., Cold Spring HarborSymp., Quant., Biol., 49, 779-787, (1984)) using the lithiumtransformation procedure of Ito et al. J. Bact., 153, 163-168, (1983).Transformed yeast cells were incubated in YPD broth (Sherman, F., etal., Methods in Yeast Genetics, Cold Spring Harbour, N.Y., 1983) at 30°C. overnight prior to plating out on selective medium (YPD plus 500ug/ml G418). This allows expression of G418-resistance and increasestransformation frequency. Colonies that came up as G418^(r) were testedon minimal medium lacking leucine (YNB,Difco+glucose+histidine+uracil+tryptophan, Sherman et al., 1983) to testfor the Leu⁺ phenotype also conferred by the plasmids. Positivetransformants (G418^(r) Leu⁺) were used for expression analysis.

The vector pPIC3-TET15 was introduced into Pichia pastoris strain GS115using the sphaeroplast transformation procedure described by Cregg etal, (1985). (Cregg et al., Molecular and Cellular Biology, 5, 3376-3385(1985)) To direct integration into the host chromosomal AOX1 locus thevector was digested with Bgl II and then mixed, in the presence ofcalcium ions and polyethylene glycol, with sphaeroplasts generated byenzymatic digestion of the cell walls with zymolyase. Transformedsphaeroplasts were regenerated in osmotically buffered agarosecontaining YNB, glucose (2%), sorbitol (1%) biotin (400 μg/1) andHis-assay medium (Difco). Transformed cells were tested for growth onmethanol since those disrupted at AOX1 by insertion of the vector shouldgrow slowly on methanol.

EXAMPLE 8

Galactose induction and preparation of cell lysates

Transformants were grown to the mid-logarithmic stage (10⁷ cells/ml) inYP broth containing 2% raffinose and 500 μg/ml G418 at 30° C. in anorbital shaker. An aliquot of 40% galactose was then added to a finalconcentration of 2% and the culture was incubated for a further 24h. Thecells were harvested by low speed centrifugation, washed once indistilled water, and resuspended in ice-cold break buffer (20 mM sodiumphosphate pH7.0, 0.1% triton X-100, 4 mM phenylmethyl sulphonylfluoride, 4 mM EGTA, and 2μg/ml each of pepstatin, antipain, leupeptinand chymostatin; 5ml for cells from a 250ml culture). Acid-washed glassbeads (0.45mm) were added and the cells were broken by vigorousvortexing. In order to remove insoluble proteins, the crude cell lysatecould be cleared by centrifugation for 15 min at 10,000 g. The proteinconcentration of the extracts was determined using the BioRad proteinassay (BioRad, according to manufacturer's instructions) and thematerial was stored at -70°.

EXAMPLE 9

Methanol Induction of Pichia cultures.

Transformants were grown at 30° C. overnight to saturation in liquidminimal medium (YNB containing biotin, 400 μg/l, and glycerol, 2% v/v).lml aliquots of these cultures were used to inoculate shake flaskscontaining 10 mls of the same medium plus 1% casamino acids. After 6-8hrs incubation at 30° C. the cells were harvested by centrifugation andresuspended in YNB (Difco) containing biotin (400 μg/l) casamino acids(1%), and methanol (0.5% v/v). After further incubation for 2-6 days thecells were harvested and lysates prepared as described for Saccharomyces(see Example 8).

Production of fragment C by high cell density Pichia pastoris cultureswas carried out using a 21 Braun fermenter equipped with monitors andcontrols for pH, dissolved O₂, stirring speed, temperature and air flow.A 10ml YNB+biotin+2% glycerol overnight culture was used to inoculatethe fermenter containing 1 litre of 5X basal salts (phosphoric acid, 42mls/l; calcium sulphate.2H₂ O. 1.8 g/l; potassium sulphate, 28.6 g/l;magnesium sulphate.7H₂ O, 23.4 g/l; potassium hydroxide, 6.5 g/l) with4mls of PTM₁ salts (cupric sulphate.5H₂ O, 6 g/l; potassium iodide, 0.08g/l; manganese sulphate.H₂ O, 3 g/l; sodium molybdate, 0.2 g/l; boricacid, 0.02 g/l; cobalt chloride, 0.5 g/l; zinc chloride, 20 g/l; ferroussulphate.7H₂ O, 65 g/l; biotin, 0.2 g/l; sulphuric acid 5 mls/l) and 5%(v/v) glycerol at 30° C. Dissolved O.sub. 2 was maintained above 20% byadjusting aeration and agitation, and the pH was maintained at pH5.0 bythe addition of 50% (v/v) ammonium hydroxide. Growth was continued untilthe glycerol was exhausted (24-30 hrs). A limited glycerol feed(containing 50% w/v glycerol and 12 ml/l PTM₁ salts) was then initiatedat 12 mls/hr for 17-21 hrs. After this period the culture was induced byreplacing the glycerol feed with a methanol feed (100% methanol plus 12ml/l PTM₁ salts) at 1 ml/hr. for 2 hrs. Then the methanol feed rate wasgradually increased over a period of 6 hours to 6 mls/hr and thefermentation was continued using these conditions for a further 46-92hrs.

EXAMPLE 10

Concentration of culture supernatants and glycoprotein analysis

Cells were induced and then harvested by centrifugation as described inExample 8. Culture supernatants were concentrated by ultrafiltrationusing Centricon 30 microconcentrators (Amicon), centrifuging at 4,000gfor 45'. Supernatants from larger scale cultures were concentrated byultrafiltration with Amicon PM30 membranes using a stirred cell.N-linked oligosaccharides were removed by digestion of concentratedsupernatants with Endoglycosidase H (Endo H, Boehringer Mannhelm).Aliquots (25 μl) were taken and 5 μl of digestion buffer added (0.2MNaH₂ PO₄, 10 mM B-mercaptoethanol, 1% SDS). After boiling for 5 minutessamples were cooled on ice and protease inhibitors added to the samefinal concentrations as given above (Example 8). Endo H (9 mU) was addedand the samples were incubated for 18hrs at 37° C. before analysis bySDS-PAGE (Example 11).

EXAMPLE 11

SDS-polyacrylamide gel analysis of proteins

Soluble or total protein extracts from induced yeast cells wereseparated by electrophoresis in SDS-polyacrylamide gels (Laemmli, UK.,Nature, 227, 680-685, (1970)). The proteins in the gel could bevisualised by staining with Coomassie Brilliant Blue R. Alternativelythe proteins were transferred to a nitrocellulose filter and reactedwith rabbit antiserum to fragment C (isolated from C.tetani) and thengoat anti-rabbit IgG conjugated to horse-radish peroxidase followed bycolour development with hydrogen peroxide and 4-chloronaphthol (BioRad).In this way the expressed fragment C could be specifically detected.

EXAMPLE 12

Immunoassay quantitation of fragment C

A two antibody sandwich enzyme-linked immunosorbent assay (ELISA) forfragment C was developed. Purified pepsinised antibody prepared fromhorses hyperimmunised with tetanus toxoid (Hughes et al., J. Appl.Bact.38, 1974, 603-622) was used to coat flexible polyvinylchloridemicrotitre plates (Dynatech, Billinghurst, GB). Coating was performedovernight at 40° C. in 50 mM sodium carbonate pH 9.5 (12.5 μg pepsinisedantibody per ml, 100 μl per well). Plates were washed three times withphosphate buffered saline pH 7.2, 0.12 (w/v) Tween (Trade Mark) 20(PBS-Tween).

Non-specific binding was reduced by incubation of plates with PBS-Tweencontaining 12% (wt/vol) BSA or 5% (wt/vol) non-fat dry milk. Plates werethen incubated sequentially with antigen, second antibody and anti-ratIgG peroxidase conjugate (Kinkegaard and Perry, Maryland, US) for 1 hrat 37° C. in each case. The second antibody was a rat monoclonal (TTO9)which binds with high affinity to fragment C (Sheppard et al., Infec.Immun. 43, 1984, 710-714) and it was used at a concentration of 2 μg/ml.

Anti-rat peroxidase conjugate was used at a dilution of 1:3000. Eachreagent was diluted into PBS-Tween containing 5% non-fat dry milk priorto addition to plates. Plates were washed three times with PBS-Tweenafter each incubation. Bound enzyme complex was assayed usingtetramethylbenzidine (TMB) as the chromogenic substrate. 1 mg TMBtablets (Wellcome Diagnostics) were dissolved in 10 ml of 0.0625Mtrisodium citrate containing 0.01% hydrogen peroxide. 100 μl of reagentwas added to each well and the reaction terminated by addition of 100 μl2M H₂ SO₄ after incubation for 10-15 mins at room temperature. Plateswere read using a Titertek multiscan plate reader (MCC/340) at 450 nm.

For quantitation, fragment C prepared from C.tetani (Fairweather, N., etal., J.Bact., 165, 21-27, (1986)) was used as a standard. Severaldilutions of this fragment C gave a narrow range titration curve with alinear portion between about 1 μl/ml and 10 μg/ml. Titration curves forthe recombinant fragment C were comparable, with similar slope and rangeto the standard curve. The titre of unknown samples were determinedeither by comparing the midpoints of titration curves or more routinelyby reading directly from the linear portion of the standard curve.

Protein was determined using the BCA assay reagent (Pierce) with bovineserum albumin as protein standard.

EXAMPLE 13

Preparation and analysis of RNA from yeast cells

In order to prepare RNA, transformed yeast cells were grown in YP+2%raffinose+500 μg/ml G418 to a density of approximately 5×10⁵ cells/ml,then induced for 24 hr after addition of 2% galactose. Total RNA wasthen prepared from the cells and analysed by Northern blotting asdescribed previously (Romanos, M. A., and Boyd, A., Nucl. Acids Res.,16, 7333-7350, (1988)). The Northern blots were probed with fragments ofpTETtac2 DNA, and its derivatives, labelled by the random priming method(Feinberg, A., and Vogelstein, B., Anal. Biochem, 132, 6-13, (1983)).

EXAMPLE 14

Immunisation of mice

Fragment C was purified from lysates of induced cells harbouringpWYG5-TET15 by affinity chromatography using TTO8 monoclonal antibody(Sheppard, A. J., et al., Infect. Immun., 43, 710-714 (1984)) linked tocyanogen bromide - activated sepharose 4B. Fragment C was eluted with0.1M sodium citrate pH3.0 and neutralised by addition of one volume of0.1M sodium phosphate pH7.0. Secreted fragment C was prepared byconcentration of supernatants from induced cultures harbouringpWYG59-TET15 without further purification.

Vaccines containing fragment C were prepared with 10% Alhydrogel (trademark) and serial dilutions were prepared. Balb/C mice were injected with0.5ml then challenged four weeks later with 100 LD₅₀ of tetanus toxin.Survivors were counted after a further four days. The results,summarised in the table below, show that yeast intracellular fragment Chas at least equal potency to E.coli produced material, whereas thesecreted fragment C is inactive.

    ______________________________________                                                        Concentration of                                              Vaccine         fragment C    Survivors                                       ______________________________________                                        1.   Yeast intracellular                                                                          50       μg/ml                                                                             5                                              fragment C     12.5     μg/ml                                                                             5                                                             3.125    μg/ml                                                                             5                                                             0.78     μg/ml                                                                             4                                         2.   Yeast secreted 50       μg/ml                                                                             0                                              fragment C     12.5     μg/ml                                                                             0                                                             3.125    μg/ml                                                                             0                                                             0.78            0                                         3.   E. coli fragment                                                                             25       μg/ml                                                                             3                                              C              6.25     μg/ml                                                                             1                                                             1.5      μg/ml                                                                             0                                         4.   PBS            0               0                                              (negative control)                                                       ______________________________________                                    

EXAMPLE 15

Immunisation of mice (also using secreted de-glycosylated material)

Fragment C was purified from lysates of induced cells harbouringpWYG5-TET15 by affinity chromatography using TTO8 monoclonal antibody(Sheppard, A. J., et al., Infect. Immun., 43, 710-714 (1984)) linked tocyanogen bromide - activated sepharose 4B. Fragment C was eluted with0.1M sodium citrate pH3.0 and neutralised by addition of one volume of0.1M sodium phosphate pH7.0. Secreted fragment C was prepared byconcentration of supernatants from induced cultures harbouringpWYG59-TET15 without further purification. To de-glycosylate thismaterial for immunisation experiments the concentrate was treated withEndott as described in Example 10 except without the addition ofmercaptoethanol and SDS and without boiling the sample.

Vaccines containing fragment C were prepared with 10% Alhydrogel (trademark) and serial dilutions were prepared. Balb/C mice were injected with0.5ml then challenged four weeks later with 100 LD₅₀ of tetanus toxin.Survivors were counted after a further four days. The results,summarised in the table below, show that yeast intracellular fragment Cwas at least as effective as the E.coli derived material, which haspreviously been shown to be equivalent to C.tetani fragment C (Makoff etal., 1989a). In contrast, the secreted product was totally inactive.However, de-glycosylation of secreted fragement C with endo H renderedit as protective as the intracellular product, syggesting that importantneutralising epitopes were masked by carbohydrate side-chains in theglycosylated form.

    ______________________________________                                        Protection of immunised mice against tetanus toxin challenge                               Survivors after challenge.sup.a                                  Antigen (dose in μg)                                                                      2       0.5   0.125  0.03  0.008                               ______________________________________                                        E. coli fragment C                                                                           .sup. 4.sup.b                                                                         2     0      0     0                                   Yeast intracellular                                                                          5       5     5      0     0                                   Yeast secreted 0       0     0      0     0                                   Yeast secreted, de-                                                                          5       5     3      0     0                                   glycosylated                                                                  Saline         0                                                              ______________________________________                                         .sup.a Groups of five mice were challenged with 100 LD.sub.50 of tetanus      toxin.                                                                        .sup.b Four mice in this group.                                          

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 13                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1359 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Clostridium tetani                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1356                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGAAAAATCTGGATTGTTGGGTTGATAATGAAGAAGATATAGATGTT48                            MetLysAsnLeuAspCysTrpValAspAsnGluGl uAspIleAspVal                             151015                                                                        ATATTAAAAAAGAGTACAATTTTAAATTTAGATATTAATAATGATATT96                            IleLeuLysLysSerThrIleLeuAsnLeuAspIle AsnAsnAspIle                             202530                                                                        AATATCGATATATCTGGGTTTAATTCATCTGTAATAACATATCCAGAT144                           AsnIleAspIleSerGlyPheAsnSerSerValIleThr TyrProAsp                             354045                                                                        GCTCAATTGGTGCCCGGAATAAATGGCAAAGCAATACATTTAGTAAAC192                           AlaGlnLeuValProGlyIleAsnGlyLysAlaIleHisLeuValA sn                             505560                                                                        AATGAATCTTCTGAAGTTATAGTGCATAAAGCTATGGATATTGAATAT240                           AsnGluSerSerGluValIleValHisLysAlaMetAspIleGluTyr                              65 707580                                                                     AATGATATGTTTAATAATTTTACCGTTAGCTTTTGGTTGAGGGTTCCT288                           AsnAspMetPheAsnAsnPheThrValSerPheTrpLeuArgValPro                               859095                                                                       AAAGTATCTGCTAGTCATTTAGAACAATATGGCACAAATGAGTATTCA336                           LysValSerAlaSerHisLeuGluGlnTyrGlyThrAsnGluTyrSer                               100105110                                                                    ATAATTAGCTCTATGAAAAAACATAGTCTATCAATAGGATCTGGTTGG384                           IleIleSerSerMetLysLysHisSerLeuSerIleGlySerGlyTrp                              115 120125                                                                    AGTGTATCACTTAAAGGTAATAACTTAATATGGACTTTAAAAGATTCC432                           SerValSerLeuLysGlyAsnAsnLeuIleTrpThrLeuLysAspSer                              130 135140                                                                    GCGGGAGAAGTTAGACAAATAACTTTTAGGGATTTACCTGATAAATTT480                           AlaGlyGluValArgGlnIleThrPheArgAspLeuProAspLysPhe                              145150 155160                                                                 AATGCTTATTTAGCAAATAAATGGGTTTTTATAACTATTACTAATGAT528                           AsnAlaTyrLeuAlaAsnLysTrpValPheIleThrIleThrAsnAsp                              165 170175                                                                    AGATTATCTTCTGCTAATTTGTATATAAATGGAGTACTTATGGGAAGT576                           ArgLeuSerSerAlaAsnLeuTyrIleAsnGlyValLeuMetGlySer                              180 185190                                                                    GCAGAAATTACTGGTTTAGGAGCTATTAGAGAGGATAATAATATAACA624                           AlaGluIleThrGlyLeuGlyAlaIleArgGluAspAsnAsnIleThr                              195200 205                                                                    TTAAAACTAGATAGATGTAATAATAATAATCAATACGTTTCTATTGAT672                           LeuLysLeuAspArgCysAsnAsnAsnAsnGlnTyrValSerIleAsp                              21021522 0                                                                    AAATTTAGGATATTTTGCAAAGCATTAAATCCAAAAGAGATTGAAAAA720                           LysPheArgIlePheCysLysAlaLeuAsnProLysGluIleGluLys                              225230235 240                                                                 TTATACACAAGTTATTTATCTATAACCTTTTTAAGAGACTTCTGGGGA768                           LeuTyrThrSerTyrLeuSerIleThrPheLeuArgAspPheTrpGly                              245250 255                                                                    AACCCTTTACGATATGATACAGAATATTATTTAATACCAGTAGCTTCT816                           AsnProLeuArgTyrAspThrGluTyrTyrLeuIleProValAlaSer                              260265270                                                                      AGTTCTAAAGATGTTCAATTGAAAAATATAACAGATTATATGTATTTG864                          SerSerLysAspValGlnLeuLysAsnIleThrAspTyrMetTyrLeu                              275280285                                                                     ACAAAT GCGCCATCGTATACTAACGGAAAATTGAATATATATTATAGA912                          ThrAsnAlaProSerTyrThrAsnGlyLysLeuAsnIleTyrTyrArg                              290295300                                                                     AGGTTATATAATGGAC TAAAATTTATTATAAAAAGATATACACCTAAT960                          ArgLeuTyrAsnGlyLeuLysPheIleIleLysArgTyrThrProAsn                              305310315320                                                                  AATGAAATAGATTC TTTTGTTAAATCAGGTGATTTTATTAAATTATAT1008                         AsnGluIleAspSerPheValLysSerGlyAspPheIleLysLeuTyr                              325330335                                                                     GTATCATATAACAAT AATGAGCACATTGTAGGTTATCCGAAAGATGGA1056                         ValSerTyrAsnAsnAsnGluHisIleValGlyTyrProLysAspGly                              340345350                                                                     AATGCCTTTAATAATCTT GATAGAATTCTAAGAGTAGGTTATAATGCC1104                         AsnAlaPheAsnAsnLeuAspArgIleLeuArgValGlyTyrAsnAla                              355360365                                                                     CCAGGTATCCCTCTTTATAAAAAAA TGGAAGCAGTAAAATTGCGTGAT1152                         ProGlyIleProLeuTyrLysLysMetGluAlaValLysLeuArgAsp                              370375380                                                                     TTAAAAACCTATTCTGTACAACTTAAATTATATGA TGATAAAAATGCA1200                         LeuLysThrTyrSerValGlnLeuLysLeuTyrAspAspLysAsnAla                              385390395400                                                                  TCTTTAGGACTAGTAGGTACCCATAATGGTCAA ATAGGCAACGATCCA1248                         SerLeuGlyLeuValGlyThrHisAsnGlyGlnIleGlyAsnAspPro                              405410415                                                                     AATAGGGATATATTAATTGCAAGCAACTGGTAC TTTAATCATTTAAAA1296                         AsnArgAspIleLeuIleAlaSerAsnTrpTyrPheAsnHisLeuLys                              420425430                                                                     GATAAAATTTTAGGATGTGATTGGTACTTTGTACCTA CAGATGAAGGA1344                         AspLysIleLeuGlyCysAspTrpTyrPheValProThrAspGluGly                              435440445                                                                     TGGACAAATGATTAA 1359                                                          TrpThrAsnAsp                                                                  450                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 452 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetLysAsnLeuAspCysTrpValAspAsnGluGluAs pIleAspVal                             151015                                                                        IleLeuLysLysSerThrIleLeuAsnLeuAspIleAsnAsnAspIle                              202530                                                                         AsnIleAspIleSerGlyPheAsnSerSerValIleThrTyrProAsp                             354045                                                                        AlaGlnLeuValProGlyIleAsnGlyLysAlaIleHisLeuValAsn                              50 5560                                                                       AsnGluSerSerGluValIleValHisLysAlaMetAspIleGluTyr                              65707580                                                                      AsnAspMetPheAsnAsnPheThrVal SerPheTrpLeuArgValPro                             859095                                                                        LysValSerAlaSerHisLeuGluGlnTyrGlyThrAsnGluTyrSer                              100105 110                                                                    IleIleSerSerMetLysLysHisSerLeuSerIleGlySerGlyTrp                              115120125                                                                     SerValSerLeuLysGlyAsnAsnLeuIleTrpThrLeuLysAspSer                               130135140                                                                    AlaGlyGluValArgGlnIleThrPheArgAspLeuProAspLysPhe                              145150155160                                                                  AsnAlaTyrLeuAla AsnLysTrpValPheIleThrIleThrAsnAsp                             165170175                                                                     ArgLeuSerSerAlaAsnLeuTyrIleAsnGlyValLeuMetGlySer                              180 185190                                                                    AlaGluIleThrGlyLeuGlyAlaIleArgGluAspAsnAsnIleThr                              195200205                                                                     LeuLysLeuAspArgCysAsnAsnAsnAsnGlnTyrVal SerIleAsp                             210215220                                                                     LysPheArgIlePheCysLysAlaLeuAsnProLysGluIleGluLys                              225230235240                                                                  LeuT yrThrSerTyrLeuSerIleThrPheLeuArgAspPheTrpGly                             245250255                                                                     AsnProLeuArgTyrAspThrGluTyrTyrLeuIleProValAlaSer                              2 60265270                                                                    SerSerLysAspValGlnLeuLysAsnIleThrAspTyrMetTyrLeu                              275280285                                                                     ThrAsnAlaProSerTyrThrAsnGly LysLeuAsnIleTyrTyrArg                             290295300                                                                     ArgLeuTyrAsnGlyLeuLysPheIleIleLysArgTyrThrProAsn                              305310315 320                                                                 AsnGluIleAspSerPheValLysSerGlyAspPheIleLysLeuTyr                              325330335                                                                     ValSerTyrAsnAsnAsnGluHisIleValGlyTyrProLysAspGly                              340345350                                                                     AsnAlaPheAsnAsnLeuAspArgIleLeuArgValGlyTyrAsnAla                              355360365                                                                     ProGlyIleProLeuT yrLysLysMetGluAlaValLysLeuArgAsp                             370375380                                                                     LeuLysThrTyrSerValGlnLeuLysLeuTyrAspAspLysAsnAla                              3853903 95400                                                                 SerLeuGlyLeuValGlyThrHisAsnGlyGlnIleGlyAsnAspPro                              405410415                                                                     AsnArgAspIleLeuIleAlaSerAsnTrpTyrPhe AsnHisLeuLys                             420425430                                                                     AspLysIleLeuGlyCysAspTrpTyrPheValProThrAspGluGly                              435440445                                                                     TrpTh rAsnAsp                                                                 450                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1359 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Clostridium tetani                                              (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                            (B) LOCATION: 1..1356                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGAAAAACCTTGATTGTTGGGTCGACAACGAAGAAGACATCGATGTT48                            MetLysAsnLeuAspCysTrpValAspAsnGluGluAspIleAspVal                              1 51015                                                                       ATCCTGAAAAAGTCTACCATTCTGAACTTGGACATCAACAACGATATT96                            IleLeuLysLysSerThrIleLeuAsnLeuAspIleAsnAsnAspIle                              20 2530                                                                       ATCTCCGACATCTCTGGTTTCAACTCCTCTGTTATCACATATCCAGAT144                           IleSerAspIleSerGlyPheAsnSerSerValIleThrTyrProAsp                              35 4045                                                                       GCTCAATTGGTGCCGGGCATCAACGGCAAAGCTATCCACCTGGTTAAC192                           AlaGlnLeuValProGlyIleAsnGlyLysAlaIleHisLeuValAsn                              5055 60                                                                       AACGAATCTTCTGAAGTTATCGTGCACAAGGCCATGGACATCGAATAC240                           AsnGluSerSerGluValIleValHisLysAlaMetAspIleGluTyr                              657075 80                                                                     AACGACATGTTCAACAACTTCACCGTTAGCTTCTGGCTGCGCGTTCCG288                           AsnAspMetPheAsnAsnPheThrValSerPheTrpLeuArgValPro                              8590 95                                                                       AAAGTTTCTGCTTCCCACCTGGAACAGTACGGCACTAACGAGTACTCC336                           LysValSerAlaSerHisLeuGluGlnTyrGlyThrAsnGluTyrSer                              100105110                                                                     ATCATCAGCTCTATGAAGAAACACTCCCTGTCCATCGGCTCTGGTTGG384                           IleIleSerSerMetLysLysHisSerLeuSerIleGlySerGlyTrp                              115120125                                                                     TCTGTT TCCCTGAAGGGTAACAACCTGATCTGGACTCTGAAAGACTCC432                          SerValSerLeuLysGlyAsnAsnLeuIleTrpThrLeuLysAspSer                              130135140                                                                     GCGGGCGAAGTTCGTCAG ATCACTTTCCGCGACCTGCCGGACAAGTTC480                          AlaGlyGluValArgGlnIleThrPheArgAspLeuProAspLysPhe                              145150155160                                                                  AACGCGTACCTGGCTAA CAAATGGGTTTTCATCACTATCACTAACGAT528                          AsnAlaTyrLeuAlaAsnLysTrpValPheIleThrIleThrAsnAsp                              165170175                                                                     CGTCTGTCTTCTGCTAACC TGTACATCAACGGCGTTCTGATGGGCTCC576                          ArgLeuSerSerAlaAsnLeuTyrIleAsnGlyValLeuMetGlySer                              180185190                                                                     GCTGAAATCACTGGTCTGGGCGCT ATCCGTGAGGACAACAACATCACT624                          AlaGluIleThrGlyLeuGlyAlaIleArgGluAspAsnAsnIleThr                              195200205                                                                     CTTAAGCTGGACCGTTGCAACAACAACAACCAG TACGTATCCATCGAC672                          LeuLysLeuAspArgCysAsnAsnAsnAsnGlnTyrValSerIleAsp                              210215220                                                                     AAGTTCCGTATCTTCTGCAAAGCACTGAACCCGAAAGAGATCGA AAAA720                          LysPheArgIlePheCysLysAlaLeuAsnProLysGluIleGluLys                              225230235240                                                                  CTGTATACCAGCTACCTGTCTATCACCTTCCTGCGTGACTTCT GGGGT768                          LeuTyrThrSerTyrLeuSerIleThrPheLeuArgAspPheTrpGly                              245250255                                                                     AACCCGCTGCGTTACGACACCGAATATTACCTGATCCCGGTAGCT TCT816                          AsnProLeuArgTyrAspThrGluTyrTyrLeuIleProValAlaSer                              260265270                                                                     AGCTCTAAAGACGTTCAGCTGAAAAACATCACTGACTACATGTACCTG86 4                          SerSerLysAspValGlnLeuLysAsnIleThrAspTyrMetTyrLeu                              275280285                                                                     ACCAACGCGCCGTCCTACACTAACGGTAAACTGAACATCTACTACCGA912                           ThrAsn AlaProSerTyrThrAsnGlyLysLeuAsnIleTyrTyrArg                             290295300                                                                     CGTCTGTACAACGGCCTGAAATTCATCATCAAACGCTACACTCCGAAC960                           ArgLeuTyrAsnGlyLeu LysPheIleIleLysArgTyrThrProAsn                             305310315320                                                                  AACGAAATCGATTCTTTCGTTAAATCTGGTGACTTCATCAAACTGTAC1008                          AsnGluIleAspSerP heValLysSerGlyAspPheIleLysLeuTyr                             325330335                                                                     GTTTCTTACAACAACAACGAACACATCGTTGGTTACCCGAAAGACGGT1056                          ValSerTyrAsnAsnAsn GluHisIleValGlyTyrProLysAspGly                             340345350                                                                     AACGCTTTCAACAACCTGGACAGAATTCTGCGTGTTGGTTACAACGCT1104                          AsnAlaPheAsnAsnLeuAspA rgIleLeuArgValGlyTyrAsnAla                             355360365                                                                     CCGGGTATCCCGCTGTACAAAAAAATGGAAGCTGTTAAACTGCGTGAC1152                          ProGlyIleProLeuTyrLysLysMetGlu AlaValLysLeuArgAsp                             370375380                                                                     CTGAAAACCTACTCTGTTCAGCTGAAACTGTACGACGACAAAAACGCT1200                          LeuLysThrTyrSerValGlnLeuLysLeuTyrAspAspL ysAsnAla                             385390395400                                                                  TCTCTGGGTCTGGTTGGTACCCACAACGGTCAGATCGGTAACGACCCG1248                          SerLeuGlyLeuValGlyThrHisAsnGlyGlnIleGly AsnAspPro                             405410415                                                                     AACCGTGACATCCTGATCGCTTCTAACTGGTACTTCAACCACCTGAAA1296                          AsnArgAspIleLeuIleAlaSerAsnTrpTyrPheAsnH isLeuLys                             420425430                                                                     GACAAAATCCTGGGTTGCGACTGGTACTTCGTTCCGACCGATGAAGGT1344                          AspLysIleLeuGlyCysAspTrpTyrPheValProThrAspGlu Gly                             435440445                                                                     TGGACCAACGACTAA1359                                                           TrpThrAsnAsp                                                                  450                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 452 amino acids                                                    (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetLysAsnLeuAspCysTrpValAspAsnGluGluAspIleAspVal                              151015                                                                        IleLe uLysLysSerThrIleLeuAsnLeuAspIleAsnAsnAspIle                             202530                                                                        IleSerAspIleSerGlyPheAsnSerSerValIleThrTyrProAsp                              35 4045                                                                       AlaGlnLeuValProGlyIleAsnGlyLysAlaIleHisLeuValAsn                              505560                                                                        AsnGluSerSerGluValIleValHisLysAlaMetA spIleGluTyr                             65707580                                                                      AsnAspMetPheAsnAsnPheThrValSerPheTrpLeuArgValPro                              8590 95                                                                       LysValSerAlaSerHisLeuGluGlnTyrGlyThrAsnGluTyrSer                              100105110                                                                     IleIleSerSerMetLysLysHisSerLeuSerIleGlySerGlyTrp                               115120125                                                                    SerValSerLeuLysGlyAsnAsnLeuIleTrpThrLeuLysAspSer                              130135140                                                                     AlaGlyGluValArgGlnIleThrP heArgAspLeuProAspLysPhe                             145150155160                                                                  AsnAlaTyrLeuAlaAsnLysTrpValPheIleThrIleThrAsnAsp                              1651 70175                                                                    ArgLeuSerSerAlaAsnLeuTyrIleAsnGlyValLeuMetGlySer                              180185190                                                                     AlaGluIleThrGlyLeuGlyAlaIleArgGluAspAsn AsnIleThr                             195200205                                                                     LeuLysLeuAspArgCysAsnAsnAsnAsnGlnTyrValSerIleAsp                              210215220                                                                     LysPheArgIlePh eCysLysAlaLeuAsnProLysGluIleGluLys                             225230235240                                                                  LeuTyrThrSerTyrLeuSerIleThrPheLeuArgAspPheTrpGly                              245 250255                                                                    AsnProLeuArgTyrAspThrGluTyrTyrLeuIleProValAlaSer                              260265270                                                                     SerSerLysAspValGlnLeuLysAsnI leThrAspTyrMetTyrLeu                             275280285                                                                     ThrAsnAlaProSerTyrThrAsnGlyLysLeuAsnIleTyrTyrArg                              290295300                                                                     Arg LeuTyrAsnGlyLeuLysPheIleIleLysArgTyrThrProAsn                             305310315320                                                                  AsnGluIleAspSerPheValLysSerGlyAspPheIleLysLeuTyr                               325330335                                                                    ValSerTyrAsnAsnAsnGluHisIleValGlyTyrProLysAspGly                              340345350                                                                     AsnAlaPheAsnAsnL euAspArgIleLeuArgValGlyTyrAsnAla                             355360365                                                                     ProGlyIleProLeuTyrLysLysMetGluAlaValLysLeuArgAsp                              370375 380                                                                    LeuLysThrTyrSerValGlnLeuLysLeuTyrAspAspLysAsnAla                              385390395400                                                                  SerLeuGlyLeuValGlyThrHisAsnGlyGlnIleGlyAsn AspPro                             405410415                                                                     AsnArgAspIleLeuIleAlaSerAsnTrpTyrPheAsnHisLeuLys                              420425430                                                                     AspLy sIleLeuGlyCysAspTrpTyrPheValProThrAspGluGly                             435440445                                                                     TrpThrAsnAsp                                                                  450                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 310 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Clostridium tetani                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTCGAGACGTCTATACTTCGGAGCACTGTTGAGCGAAGGCTCATTAGATATATTTTCTGT60                CATTT TCCTTAACCCAAAAATAAGGGAGAGGGTCCAAAAAGCGCTCGGACAACTGTTGAC120              CGTGATCCGAAGGACTGGCTATACAGTGTTCACAAAATAGCCAAGCTGAAAATAATGTGT180               AGCCTTTAGCTATGTTCAGTTAGTTTGGCTAGCAAAGATATAAAAGCAGGTCGGAA ATAT240              TTATGGGCATTATTATGCAGAGGATCCACATGATAAAAAAAACAGTTGAATATTCCCTCA300               AAAATGACTG310                                                                 (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Synthetic                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: /function=sticky ends                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GATCTAAACGATGAAAAACCTTGATTGTTGGG32                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: synthetic                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: /function=sticky ends                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TCGACCCAACAATCAAGGTTTTTCATCGTTTA32                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 63 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: SYNTHETIC                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: /function="STICKY END"                                 /product="LABEL"                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GATCTACATGATAAAAAAAAGAGTTGAATATTCCCTCAACCATGGTTAACTTGGACTGTT60                GGG63                                                                         (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 63 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: SYNTHETIC                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..4                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                      TCGACCCAACAGTCCAAGTTAACCATGGTTGAGGGAATATTCAACTGTTTTTTTTATCAT60                GTA63                                                                         (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 312 base pairs                                                     (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: synthetic                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 42..311                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GATCTACATGATAAAAAAAACAGTTGAATATTCCCTCA AAAATGAGATTTCCT53                      MetArgPhePro                                                                  TCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTA GCTGCT101                          SerIlePheThrAlaValLeuPheAlaAlaSerSerAlaLeuAlaAla                              5101520                                                                       CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCG GCTGAA149                          ProValAsnThrThrThrGluAspGluThrAlaGlnIleProAlaGlu                              253035                                                                        GCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGC TGTT197                          AlaValIleGlyTyrSerAspLeuGluGlyAspPheAspValAlaVal                              404550                                                                        TTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACT 245                          LeuProPheSerAsnSerThrAsnAsnGlyLeuLeuPheIleAsnThr                              556065                                                                        ACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA293                           ThrIl eAlaSerIleAlaAlaLysGluGluGlyValSerLeuGluLys                             707580                                                                        AGAGAGGCTGAAGCCATGG312                                                        ArgGluAlaGluAlaM et                                                           8590                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      MetArgPheProSerIlePheThrAlaValLeuPheAlaAlaSerS er                             151015                                                                        AlaLeuAlaAlaProValAsnThrThrThrGluAspGluThrAlaGln                              202530                                                                        IleProAla GluAlaValIleGlyTyrSerAspLeuGluGlyAspPhe                             354045                                                                        AspValAlaValLeuProPheSerAsnSerThrAsnAsnGlyLeuLeu                              5055 60                                                                       PheIleAsnThrThrIleAlaSerIleAlaAlaLysGluGluGlyVal                              65707580                                                                      SerLeuGluLysArgGluAlaGluAlaMet                                                 8590                                                                         (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 75 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: synthetic                                                       (ix) FEATURE:                                                                 ( A) NAME/KEY: miscfeature                                                    (B) LOCATION: 1..2                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CGAAGGATCCAAACGATGAGATTTCCTTCAATTTTTACTGCAGACTAGTCCCGGGTAAGT60                AAGTAAGCGGCCGCG75                                                             (2) INFORMATION FOR SEQ ID NO:13:                                             ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 77 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: synthetic                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: miscfeature                                                     (B) LOCATION: 1..4                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AATTCGCGGCCGCT TACTTACTTACCCGGGACTAGTCTGCAGTAAAAATTGAAGGAAATC60               TCATCGTTTGGATCCTT77                                                       

What we claim is:
 1. An expression vector which incorporates DNAencoding tetanus toxin fragment C having the amino acid sequence shownin SEO ID NO:2 and having an increased (G+C)-content relative to thewild-type DNA sequence show in SEO ID NO:1 in each of the followingregions:(i) from nucleotide 510 to nucleotide 710, (ii) from nucleotide650 to nucleotide 850, (iii) from nuceotide 800 to nucleotide 1100, (iv)from nucleotide 900 to nucleotide 1200 and, (v) from nucleotide 1100 tonucleotide 1356.the numbers corresponding to those set forth in thesequence of SEO ID NO:1 so as to allow the production of complete mRNAtranscripts in yeast, which vector thereby expresses said fragment C inyeast, wherein the said DNA is as shown in SEQ ID NO:3.
 2. A yeastorganism transformed with an expression vector which incorporates DNAencoding tetanus toxin fragment C having the amino acid sequence shownin SEO ID NO:2 and having an increased (G+C) -content relative to thewild-type DNA sequence shown in SEO ID NO:1 in each of the followingregions:(i) from nucleotide 510 to nucleotide 710, (ii) from nucleotide650 to nucleotide 850, (iii) from nucleotide 800 to nucleotide 1100,(iv) from nucleotide 900 to nucleotide 1200 and, (v) from nucleotide1100 to Nucleotide 1356, the numbers corresponding to those set forth inthe sequence of SEO ID NO:1, so as to allow the production of completemRNA transcripts in yeast, which vector thereby expresses said fragmentC in yeast, wherein the said DNA is as shown in SEQ ID NO:3.
 3. Aprocess for the preparation of fragment C of tetanus toxin, whichcomprises the culturing of a yeast organism transformed with anexpression vector which incorporates DNA encoding tetanus toxin fragmentC having the amino acid sequence shown in SEO ID NO:2 and having anincreased (G+C)-content relative to the wild-type DNA sequence shown inSEO ID NO:1 in each of the following regions:(i) from nucleotide 510 tonucleotide 710, (ii) from nucleotide 650 to nucleotide 850, (iii) fromnucleotide 800 to nucleotide 1100, (iv) from nucleotide 900 tonucleotide 1200 and, (v) from nucleotide 1100 to nucleotide 1356,thenumbers corresponding to those set forth in the sequence of SEO ID NO:1,so as to allow the production of complete mRNA transcripts in yeast,which vector thereby expresses said fragment C in yeast, wherein thesaid DNA is as shown in SEQ ID NO: 3.